Regulation of gene expression is crucial for proper development. Our studies aim to understand the mechanism of epigenetic inheritance of gene expression and uncover unrecognized ways to disrupt gene expression that may contribute to developmental defects in humans. Polycomb group (PcG) and the Trithorax group (TrxG) proteins are important for epigenetic inheritance of the silenced and the active chromatin state, respectively. They are present in all metazoans. PcG proteins deposit the repressive chromatin mark H3K27me3 over PcG-regulated genes. In Drosophila, regulatory elements called Polycomb group response elements (PREs) are required for the recruitment of chromatin-modifying PcG protein complexes to the DNA. TrxG proteins act through either the same or overlapping cis-acting sequences as PcG proteins. Our group is working on understanding how PcG and TrxG proteins get recruited to the DNA. Another goal of our research is to understand how PREs interact with other regulatory elements present within a locus. During the past year, we have made important advances in understanding PcG recruitment by PREs. In addition, our recent experiments show how chromatin context influences Polycomb domain structure and function. PREs are made up of binding sites for DNA binding proteins that recruit PcG proteins complexes to chromatin. Over the years our lab has identified three PRE recruiter proteins: Pho, Spps, and Cg. Although mutation of PREs for Pho, Spps, or Cg binding sites compromises PcG recruitment/stability in transgenes, the role of these proteins in PcG complex recruitment/stability within native PcG target genes is not understood, We conducted a comparative OMICs analysis of Spps, Pho, and Cg proteins in wild-type and mutant larvae lacking each of these recruiters. The survival of Spps maternal-zygotic mutants late in development allowed us a unique opportunity to study PcG protein binding in the complete absence of a recruiter protein in an intact animal. Our comparison of the genomic distribution of three recruiter proteins, Spps, Pho, and Cg, in wild-type and mutant larvae allows us to make the following conclusions: 1. Recruitment of PcG proteins is combinatorial and redundant at many sites. The strongest recruitment of PcG proteins occurs when all three recruiter proteins are present. 2. Spps and pho mutants show a global reduction in H3K27me3 levels from canonical Polycomb domains and a redistribution of H3K27me3 to heterochromatin. 3. Loci with drastically reduced H3K27me3 levels in Spps mutants have a concomitant loss of Pho-RC, PRC1, and PRC2 components. 4. Individual PREs respond differently to the loss of one factor. 5. Spps and Pho play additional and semi-independent roles outside of H3K27me3 domains and show differential association with Psc and Ph implying the recruitment of PRC1 variants rather than the canonical PRC1 at some of the sites. 5. Global reduction in H3K27me3 levels from canonical Polycomb domains does not result in global derepression of associated genes. In summary, our results define a role for Spps in PcG recruitment and highlight resiliency and diversity of PREs and recruitment mechanisms. The combinatorial recruitment of PcG protein complexes and the diversity of PREs in Drosophila shows a level of complexity of PcG protein recruitment not previously recognized. Our data should guide studies on PcG recruitment in mammals where it is much less understood. We are interested in how PREs act with positive regulatory elements (enhancers) to regulate expression of developmental genes. In Drosophila, the invected (inv) and engrailed (en) genes form a Polycomb-regulated chromatin domain that extends about 113kb. These two genes encode highly related homeodomain proteins that are co-regulated in a complex manner throughout development. For example, in the embryo, inv/en are co-expressed in stripes, and specific cells in the head, tail, gut, CNS and PNS while in larvae, inv/en are expressed in, and required for the formation of the posterior compartment of imaginal discs. In our dissection of inv/en regulatory DNA, we found 16 discrete DNA fragments that act as enhancers in reporter constructs to recapitulate aspects of inv/en embryonic expression. In contrast, we could not find a DNA fragment that could drive expression of a reporter construct in the posterior compartment of imaginal discs. Instead we identified a 2.8kb DNA fragment that caused reporter gene expression in the anterior compartment of wing imaginal discs, the opposite of what we expected. We hypothesized that this was an imaginal disc enhancer (IDE). We generated a 79-kb HA-En transgene that could fully rescue inv/en double mutants. Deletion of the IDE from this transgene proved that this was, in fact, an enhancer for expression of inv/en in the posterior compartment of the wing imaginal discs. ChIP-seq experiments show that this IDE contains binding sites for PcG proteins as well as the En protein itself. Our data suggests that, when the IDE is outside of the inv/en domain, En binds to the IDE and silences expression in the posterior compartment, while activators directly activate the IDE in the anterior compartment. When this IDE is present within the inv/en gene complex, activating chromatin marks, epigenetically inherited from inv/en expressing embryonic cells, prevent En from repressing itself. In contrast, cells in the anterior compartment are derived from embryonic cells that do not express inv/en. H3K27me3 and PcG proteins are bound to inv/en DNA in the OFF transcriptional state. This repressive chromatin mark is inherited in cells in the anterior compartment of imaginal discs. Thus, our data show the importance of chromatin context and epigenetic memory on the activity of a developmentally important enhancer. Polycomb group proteins (PcGs) drive target gene repression and form large chromatin domains. In Drosophila, DNA-PREs recruit PcGs to the DNA. We have previously shown that within the invected-engrailed (inv-en) Polycomb domain, strong, constitutive PREs are dispensable for Polycomb domain structure and function. We suggest that the endogenous chromosomal location imparts stability to this Polycomb domain. To test this a 79kb en-transgene was inserted into other chromosomal locations. This transgene is functional and forms a Polycomb domain. The spreading of the H3K27me3 repressive mark, characteristic of PcG domains, varies depending on the chromatin context of the transgene. Unlike at the endogenous locus, deletion of the strong, constitutive PREs from the transgene leads to both loss- and gain-of function phenotypes, demonstrating the important role of these regulatory elements. Our data show that chromatin context plays an important role in Polycomb domain structure and function.